Giardia lamblia, an important human pathogen, must successfully excyst in order to infect a new host. Since excystation is too rapid to rely entirely on new gene expression, the unifying hypothesis of this proposal is that second messenger pathways may be central for regulation. While we can reproduce the entire life cycle in vitro, neither the precise stimuli nor the second messenger pathways that regulate excystation are known. Based on extensive Preliminary Data, we propose these Specific Aims: Specific Aim 1 is to define the key physiological excystation stimuli, and to test the hypothesis that Giardia cysts' responses to these signals are mediated by or reflected in changes in cytosolic calcium and/or cyclic AMP. We will ask whether pharmacological release of "caged" Ca 2v and/or cAMP into the cytosol can bypass the corresponding excystation stimulus. Specific Aim 2 is to define the role(s) of calmodulin (CAM) in regulating excystation and to identify specific "downstream" effectors that carry out its function. gCaM co-localizes with protein kinase A to the flagellar basal bodies-centrosomes, suggesting a key role in coordinating these pathways. We will use in vivo cross-linking and genomic data to functionally identify downstream effectors that specifically interact with CaM during excystation. Specific Aim 3 is to test the hypothesis that changes in protein phosphorylation may be important in regulating excystation, compared with de novo protein synthesis. We will compare changes in protein phosphorylation (the "phosphoproteome"), with overall changes in the protein expression in excysting cells. Sequence analyses and genomic data will permit us to identify key excystation proteins. This proteomic approach will test our overall hypothesis and complement the cellular and molecular data of Aims 1 and 2. Our proposed studies will reveal important insights into regulation of G lamblia excystation. Many other parasites have cystic forms whose excystation is required for transmission. Giardia may be a valuable model for understanding parasite differentiation in the intestinal tract. On a basic level, Giardia excystation is a unique model for cellular awakening from dormancy in response to environmental signals in an early-diverging eukaryotes.